Nitric oxide (NO) is critically involved in numerous functions in the developing lung. NO is produced by neuronal NO synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). We have shown that all three isoforms are upregulated in the third trimester in fetal baboon lung. Chronic Lung Disease (CLD) is a condition which disrupts preterm human lung development, resulting in airway, parenchymal and pulmonary vascular dysfunction. We have shown in the baboon model of CLD that lung nNOS and eNOS are markedly decreased, that lung NO production remains low during the early course, and that inhaled NO gas (iNO) improves pulmonary hemodynamics, compliance and resistance. iNO also partially normalizes surfactant function, it stimulates lung growth, and it prevents certain structural features of CLD. Using physiologic, biochemical and histologic/morphologic readouts, the overall objective of this proposal is to determine if interventions which upregulate endogenous NO production, or which replace NO in a manner favoring physiologic NO metabolism, result in greater prevention of CLD. Studies will be done in preterm baboons born at 125d gestation (term = 185d) and ventilated for 14d. Aim 1 is to determine the effects of postnatal estradiol (E2) treatment on lung NO production, and on the development of CLD. E2 upregulates nNOS and eNOS expression and activity in some paradigms, and with preterm birth there is withdrawal from placentally-derived E2. Aim 2 is to determine if there is greater impact on the genesis of CLD with iNO plus glutathione (GSH), or with O-nitrosoethanol (ENO), versus iNO alone. Whereas endogenous NO forms biologically active S-nitrosothiols (SNO), iNO gas yields toxic higher oxides of nitrogen (NOX) and peroxynitrite. GSH, which is deficient in prematurity, is the primary substrate for lung SNO. In contrast to iNO, ENO preferably forms SNO and generates less NOX and peroxynitrite in mature models tested thus far. Aim 3 is to delineate the changes in pulmonary and systemic NO metabolism in CLD and in the intervention groups. In initial work, blood NOX rose while SNO fell postnatally in CLD, and iNO raised NOX further with scant impact on SNO. It is hypothesized that E2, iNO + GSH, or ENO will increase SNO and cause little change in NOX and peroxynitrite. These studies will increase our knowledge of the role of NO in CLD and also test three novel, mechanistically-based therapies.